Stempro chondrogenic differentiation media

Sorted hDPSCs in centrifuge tubes were pelleted by centrifugation at 400g for 10 minutes and incubated for 1 h in basal media. Following this incubation, the media was aspirated and replaced with StemPro Chondrogenic Differentiation media (Life Technologies), cells were cultured for 2 weeks at 37 °C in 5 % CO₂ in air with media changes every 3 days. Then, the culture cells were washed twice in PBS and fixed in 10 % formalin for 10 min before further fixation in fresh 10 % formalin for 1 h. Fixed cells were washed twice with dH₂O and once with 3 % (v/v) acetic acid in dH₂O for 3 min before staining with 1 % (w/v) Alcian Blue in 3 % (v/v) acetic acid for 30 min. Samples were then washed 3 times in dH₂O and imaged.

A total of 2 × 10⁵ MSC were centrifuged at 300 × g for 5 min to form a pellet and were then cultured in Stempro Chondrogenic differentiation media (Life technologies, UK) for 21 days under standard cell culture conditions with media changes every 3–4 days in a 15 ml polypropylene falcon tube (Greiner Bio‐one, Australia). The pellet was then fixed in 4% paraformaldehyde (Sigma, UK) for 1 h before paraffin embedding, sectioning at 5 μm, staining with Alcian Blue and imaged using light microscopy.